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Method Article

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

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DOI:

10.3791/51519

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July 24th, 2014

 ,  ,  , 
1NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health

In This Article

Summary

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Abstract

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Introduction

The capacity of hPSCs to differentiate toward multilineage adult tissues has opened new avenues to treating patients who suffer from severe diseases that involve cardiovascular, hepatic, pancreatic, and neurological systems1-4. Various cell types derived from hPSCs would also provide robust cellular platforms for disease modeling, genetic engineering, drug screening, and toxicological testing1,4. The key issue that ensures their future clinical and pharmacological applications is the generation of large numbers of clinical-grade hPSCs through in vitro cell culture. However, current culture systems are either insufficient or inherently variable, involving various feeder and feeder-free cultures of hPSCs as colonies5,6.

Colony-type growth of hPSCs shares many structural features of the inner cell mass (ICM) of early mammalian embryos. The ICM is prone to differentiate into the three germ-layers in a multicellular environment because of the existence of heterogeneous signaling gradients. Thus, the acquisition of heterogeneity in early embryonic development is considered as a required process for differentiation, but an unwanted feature of hPSC culture. The heterogeneity in hPSC culture is often induced by excessive apoptotic signals and spontaneous differentiation due to suboptimal growth conditions. Thus, in colony-type culture, the heterogeneous cells are often observed in the periphery of the colonies7,8. It has been also shown that the cells in human embryonic stem cell (hESC) colonies exhibit differential responses to signaling molecules such as BMP-4 9. Moreover, colony culture methods produce low cell yields as well as very low cell recovery rates from cryopreservation due to uncontrollable growth rates and apoptotic signaling pathways6,9. In recent years, various suspension cultures have been developed for culturing hPSCs, particularly for expansion of large amounts of hPSCs in feeder- and matrix-free conditions6,10-13. Obviously, different culture systems have their own advantages and disadvantages. In general, the heterogeneous nature of hPSCs represents one of the major drawbacks in colony-type and aggregated culture methods, which are suboptimal for delivering DNA and RNA materials into hPSCs for genetic engineering6.

Clearly, there is an imperative need to develop new systems that circumvent some shortcomings of current culture methods. The discoveries of small molecule inhibitors (such as the ROCK inhibitor Y-27632 and JAK inhibitor 1) that improve single-cell survival pave the way for dissociated-hPSC culture14,15. With the use of these small molecules, we have recently developed a culture method based on non-colony type (NCM) growth of dissociated-hPSCs9. This novel culture method combines both single-cell passaging and high-density plating methods, allowing us to produce large amounts of homogeneous hPSCs under consistent growth cycles without major chromosomal abnormalities9. Alternatively, NCM culture might be implemented with different small molecules and defined matrices (such as laminins) in order to optimize the culture method for wide applications. Here, we present several detailed protocols based on NCM culture and delineate detailed procedures for genetic engineering. To demonstrate the versatility of NCM protocols, we also tested NCM culture with diverse ROCK inhibitors and with the single laminin isoform 521 (i.e., LN-521).

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Protocol

Single-cell based non-colony type monolayer (NCM) culture of hPSCs.

1. Preparations

  1. Make 500 ml of medium for culture of mouse embryonic fibroblasts (MEFs): DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, and 0.1 mM non-essential amino acids (NEAA).
  2. Isolate mouse embryonic fibroblasts (MEFs) cells derived from the CF1 strain following a routine protocol16 and culture MEFs on 0.1% gelatin-coated 6-well cell culture plate in DMEM medium. Alternatively, purchase MEF stocks at passage 3 from commercial resources.
  3. Prepare Matrigel plates.
    1. Dilute 5 ml of hESC-qualified Matrigel stock with 5 ml of DMEM/F12 medium (chilled at ~4 °C) and store as 50% aliquots in a -20 °C freezer. Thaw the frozen Matrigel stock in a refrigerator (~4 °C) O/N and further dilute the Matrigel in cold DMEM/F12 medium (to give rise to a 2.5% working concentration).
    2. Coat 6-well cell culture plates with 1.5 ml of 2.5% Matrigel per well, store the coated plates in refrigerator O/N (Note: use the Matrigel-coated plate within 2 weeks).
    3. Remove the Matrigel plate from the refrigerator, allow the plate to warm at RT in the cell culture hood for 10 to 30 min (Note: do not warm the plate in a cell culture incubator), and aspirate DMEM medium prior to plating hPSCs.
  4. Make 500 ml hESC medium: 80% DMEM/F12 medium, 20% KSR, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, and 4 ng/ml of FGF-2.
  5. Prepare 10 ml of 2X hPSC freezing medium: 60% FBS, 20% DMSO, and 20 µM Y-27632 in mTeSR1 medium, sterilize by filtration, and use the medium within 1 week.

2. Protocol 1 (Basic): Grow hPSC Colonies on Feeders

  1. Use MEF passage numbers at 5 or 6 (designated as p5 and p6) for hPSC culture in order to get consistent results. Mitotically inactivate MEFs by treating the cells with 10 μg/ml mitomycin C for 3 hr at 37 °C, wash the cells 3x with 1X Dulbecco’s Phosphate-Buffered Saline (D-PBS), and then dissociate MEFs with 0.05% Trypsin in 0.53 mM EDTA. Alternatively, irradiate MEFs at a dose of 8,000 rads with an X-ray irradiator.
  2. Count cell numbers using the Trypan Blue stain exclusion method under microscope. Alternatively, use an automatic cell counter.
  3. Plate irradiated MEFs on 6-well polystyrene plates coated with 0.1% gelatin at a density of 1.88 x 105 cells per well (i.e., 1.96 x 104 cells/cm2). Incubate the cells at 37 °C and 5% CO2 for 24 hr.
  4. Remove the MEF culture medium by aspiration with a Pasteur pipette (Note: no wash steps needed at this time).
  5. Triturate hPSC colonies from previous culture into small clumps, examine the clumps under microscope to ensure their sizes ranging from 50 to 100 μm in diameters, and plate hPSC clumps on the top of MEF feeder layers in one well containing 2 ml of hPSC medium.
  6. Change hESC medium daily for 3 to 5 days, record colony growth by photograph, mark any morphologically altered or differentiated colonies (~5%), and manually remove the marked colonies by gently aspiration with a Pasteur pipette.
  7. Rinse the remaining colonies on MEFs twice (2 min each with D-PBS), and incubate the colonies with 2 ml of 1 mg/ml collagenase IV in hPSC medium for 10 to 30 min), and examine the detachment of hPSC colonies from the MEF-coated surface (Note: use a scraper to help the detachment process only if the colonies are tightly attached to the plate).
  8. Add 5 ml of hPSC medium to each well to minimize enzymatic reactions, transfer colonies to a 15 ml tube, allow hPSC colonies to sediment for 3 to 5 min at RT, and ensure the sedimentation of colonies by direct visualization of the cells at the bottom of the tube (Note: do not centrifuge the tube at this step).
  9. Remove the supernatants containing residual MEFs and dissociated single cells, resuspend the colonies with 5 ml of hESC medium, and repeat the sedimentation step twice.
  10. Remove the medium, triturate hPSC colonies into small clumps, store in 1X hPSC freezing medium (or CryoStore CS10 freezing medium) for cryopreservation (1 confluent well per frozen vial) or plate on a 6-well plate for cell passaging.

3. Protocol 2: Convert hPSC Colonies from Feeders to NCM

  1. Rinse hPSC pellets in D-PBS once, incubate the pellets with 1 ml of 1X Accutase for 10 min, and examine the enzymatic reaction under a microscope to ensure single-cell dissociation.
  2. Terminate the enzymatic reactions by gently resuspending the cells in 5 ml of mTeSR1 medium containing 10 μM Y-27632 followed by centrifugation at 200 x g at RT for 5 min (Note, do not use excessive centrifugation forces which cause cell damage).
  3. Add 5 ml of mTeSR1 medium to the pellet, resuspend the pellet as single cells, and filter dissociated cells through a 40 µm cell strainer (to remove any residual cell aggregates).
  4. Seed 1.3-2 x 106 hPSCs into one well (1.4-2.1 x 105 cells/cm2) of a 6-well plate coated with 2.5% hESC-qualified Matrigel, add mTeSR1 medium up to 2.5 ml, and include 10 µM Y-27632 in the medium (to facilitate the initial 24 hr single-cell plating). Alternatively, utilize the following small molecules to replace 10 μM Y-27632 for enhancing single-cell plating: 1 µM Y-39983 (ROCK I inhibitor), 1 µM phenylbenzodioxane (ROCK II inhibitor), 1 µM thiazovivin (a novel ROCK inhibitor), and 2 μM JAK inhibitor 1.
  5. Replace the medium with drug-free mTeSR1 medium on the next day (within 24 hr), dissociate cells from one extra well, and count cell number to determine single-cell plating efficiency (Note: approximately, 50 to 90% of single-cell plating efficiency that can be achieved at this stage).
  6. Allow the cells to grow as a single-cell-formed monolayer for a few days with 3 ml of mTeSR1 medium. Change medium daily.
  7. Empirically determine the schedule for passaging adapted NCM depending on the cell density. Passage when cell growth reaches confluence at day 3 or day 4, or at the 4 hr time point after the disappearance of cell-to-cell borders. Dissociate the cells in 1 ml of Accutase, use a 1 to 3 splitting ratio (i.e., 1 confluent well of cells plated in 3 wells of 6-well plate) for cell passaging, and stabilize adapted NCM by 5 passages.
  8. Cell freezing
    1. Utilize one confluent well (~ 5-6 x106 cells) for one frozen vial: dissociate cells from one confluent well with 1 ml of Accutase for 10 min.
    2. Dilute with 5 ml of mTeSR1 medium and centrifuge cells as described above.
    3. Resuspend the pellet in 500 μl of mTeSR1 medium and then slowly add 500 μl of 2X hPSC freezing medium (containing 20 μM Y-27632) in a cryopreservation vial. Alternatively, resuspend the cells in 1X hPSC freezing medium containing 2 μM JAK inhibitor 1 or 1X CryoStor CS10 medium in the presence of either 10 µM Y-27632 or 2 μM JAK inhibitor 1.
    4. Place frozen vials into an ice-chilled cryocontainer (bottom-filled with isopranol) and transfer the cryocontainer to a -80 °C freezer immediately.
    5. Transfer cells from the -80 °C freezer to a liquid nitrogen tank (on the next day) for long-term cryopreservation.
  9. Cell thawing
    1. Utilize one cryopreservation vial for plating 1 well of a 6-well plate.
    2. Pre-warm mTeSR1 medium in a 37 °C water bath for 10 min, thaw cells in the same water bath for 2 min, drop-wise add cells to 5 ml of pre-warmed mTeSR1 medium containing 10 µM Y-27632, and centrifuge as described above.
    3. Gently resuspend the cell pellets with 2 ml of mTeSR1 medium containing 10 µM Y-27632 and slowly transfer cells to a well pre-coated with Matrigel (Note: avoid producing air bubbles).
    4. Change medium daily and expect hPSC growth to reach confluence at day 3 or 4.

4. Protocol 3: Convert hPSC Colonies on Matrigel to NCM Culture

  1. Remove a Matrigel-coated plate from refrigerator, place the plate in the tissue culture hood for 10 to 30 min, remove the medium from each well of the plate, and add 2 ml of pre-warmed mTeSR1 medium to each well.
  2. Transfer triturated hPSC clumps from the feeder culture (Step 2.10 of the basic protocol) to the above Matrigel plate. Add mTeSR1 medium up to 2.5 ml final volume in each well.
  3. Change medium daily for 3 to 4 days until colonies reach 80 to 90% confluence.
  4. For cell passaging: rinse the colonies with D-PBS twice, treat the cells with 1 ml of 2 mg/ml dispase at 37 °C for 15 to 20 min, and sediment and passage cells as clumps.
  5. For NCM culture: repeat Steps 3.1 to 3.9 described in Protocol 2.

5. Protocol 4: NCM Culture of hPSCs on LN-521

  1. Thaw the recombinant LN-521 solution at 4 °C before the day of use and make the laminin coating solution (LCS) by diluting the thawed laminin with 1X D-PBS (containing Ca2+/Mg2+) to a final concentration of 10 μg/ml.
  2. Add 1 ml of the LCS to one well in 6-well plate (note: avoid dry-out during the coating process).
  3. Seal the coated plate with Parafilm to prevent evaporation and store the plate in refrigerator (4 °C) O/N (note: use the plate within 1 week).
  4. Gently remove the LCS with a Pasteur pipette without touching the coated surface and add 2 ml of mTeSR1 medium to one well.
  5. Warm all culture solutions (including D-PBS) in a 37 °C water bath for 25 min.
  6. Convert hPSC colonies to NCM
    1. Dissociate cell aggregates to single cells with Accutase as described in Protocol 2.
    2. Seed 1.3-2 x 106 hPSCs into one LN-521-coated well (1.4-2.1 x 105 cells/cm2) without the presence of ROCK inhibitors.
    3. Change medium daily for 3 to 4 days.
    4. Dissociate the cells in 1 ml of Accutase at day 3 or 4 for the next cell passaging using a 1 to 3 splitting ratio.

6. Protocol 5: NCM Culture for Plasmid DNA Transfection

  1. Adapt hPSC colonies to NCM culture as described in Protocols 2 to 4.
  2. Plate dissociated hPSCs in 12-well plate with a cell density of 7.5 x 105 cells/well in mTeSR1 medium in the presence of 10 μM Y-27632.
  3. Replace mTeSR1 medium with drug-free mTeSR1 medium between 4-8 hr after plating the cells.
  4. For each transfection, dilute 2.5 μg of expression plasmids (e.g., pmaxGFP) and 5 μl of Lipofectamine 2000 in separate Eppendorf tubes in 125 μl each of Opti-MEM Reduced Serum Medium.
  5. After 5 min, mix the diluted reagents and incubate for 20 min at RT (to form transfection complexes).
  6. Add the 250 μl transfection complexes to each well containing hPSCs in 1 ml mTeSR1 medium and incubate the cells at 37 °C in a CO2 incubator for 24 hr.
  7. Examine the transfection efficiency under a fluorescence microscope and photograph randomly for calculation of the actual transfection efficiency.

7. Protocol 6: NCM Culture for Transfection of MicroRNAs

  1. Dissociate semi-confluent hPSCs at day 2 under NCM conditions by adding 1 ml of Accutase per well in 6-well plate.
  2. Add 10 ml of mTeSR1 medium to dilute enzymatic reactions and centrifuge at 200 x g for 5 min.
  3. Resuspend the cell pellet with mTeSR1 medium, seed the cells at a density of 7.5 x 105 cells per well in a 12-well plate in mTeSR1 medium containing 10 μM Y-27632, and let the cells attach for 4 hr.
  4. Replace the medium with Y27632-free mTeSR1 medium.
  5. Use commercially available microRNAs (e.g., a non-targeting miRIDIAN miRNA Transfection Control labeled with Dy547). Titrate concentrations ranging from 0-160 nM using the provided reagents and follow the manufacturers’ instructions.
  6. Optimize transfection efficiency for each concentration in hPSC lines by imaging the Dy547 fluorescence of the live cells under a microscope at 24 hr after the transfection.
  7. Calculate the transfection efficiencies based on the percentage of Dy547 positive cells over all imaged cells.

8. Protocol 7: NCM Culture for Transduction of Lentiviral Vector

  1. Repeat steps 7.1 to 7.4 of Protocol 6.
  2. Calculate the amounts of viral particles to be used for transduction: Use the following formula: TU = (MOI x CN) / VT, whereas TU denotes the total numbers of transforming units, MOI equals the desired multiplicity of infection in the well (MOI or TU/cell), CN specifies the number of cells in each well, and VT indicates stock viral titers (TU/ml). For example, given that stock viral titers for SMART-shRNA vector equals 1 x 109 TU/ml (transforming units per ml), 7.5 x 105 cells per well at the time of transduction, and an expected MOI of 20: use 15 μl of the stock viral particles for each well (note: this condition is recommended for transient lentiviral induction).
  3. Pre-warm 300 μl of mTeSR1 medium with 10 mg/ml of polybrene for 30 min.
  4. Add 15 μl of stock viral particles into the pre-warmed mTeSR1 medium containing polybrene and gently mix the solution.
  5. Replace the cell culture medium with 300 μl of prewarmed medium containing the viral particles.
  6. After 4 hr incubation, examine turboGFP fluorescence (a maker for shRNA expression) to determine the transduction efficiency.
  7. Add 300 μl of additional mTeSR1 medium to the transducing well when the cells begin to express turboGFP.
  8. After 12-16 hr incubation, reexamine turbo-GFP expression.
  9. Perform desired follow-up experiments with these transduced cells within 72 hr.

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Results

A general schema of NCM culture

Figure 1 represents a typical NCM culture schema showing the dynamic changes of hPSCs after high-density single-cell plating in the presence of the ROCK inhibitor Y-27632. These morphological changes include intercellular connections after plating, cellular clusters formation, and exponential cell growth followed by cell condensation (Figure 1A). A representative experiment indicates WA01 (H1) hESCs, plated as s...

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Discussion

There are two major ways to culture hPSCs in vitro: conventional colony-type culture (of cells on feeders or extracellular matrices) and suspension culture of hPSCs as aggregates without feeders6. The limitations of both colony-type and suspension culture methods include accumulated heterogeneity and inheritable epigenetic changes. NCM culture, based on both single-cell passaging and high-density cell plating, represents a new culture method for hPSC growth6,18. Although various single-cell...

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Disclosures

The authors declare that they have no competing financial interests.

Acknowledgements

This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) at the National Institute of Neurological Disorders and Stroke. We would like to thank Dr. Ronald D. McKay for his discussion and comments on this project.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Countess automated cell counterInvitrogen Inc.C10227Automatic cell counting
Faxitron Cabinet X-ray SystemFaxitron X-ray Corporation, Wheeling, IL Model RX-650X-ray irradiation of MEFs
MULTIWELL 6-well platesBecton Dickinson Labware353046Polystyrene plates
DMEMInvitrogen Inc.11965–092For MEF medium
Mitomycin CRoche107409Mitotic inhibitor
TrypsinInvitrogen Inc.25300-054For MEF dissociation
DMEM/F12Invitrogen Inc.11330–032For hPSC medium
Opti-MEM I Reduced Serum Medium Invitrogen Inc.31985-062For hPSC transfection
Heat-inactivated FBSInvitrogen Inc.16000–044Component of MEF medium
Knockout Serum ReplacementInvitrogen Inc.10828–028KSR, Component of hPSC medium
Dulbecco’s Phosphate-Buffered SalineInvitrogen Inc.14190-144D-PBS, free of Ca2+/Mg2+
Non-essential amino acidsInvitrogen11140–050NEAA, component of hPSC medium
L-GlutamineInvitrogen 25030–081Component of hPSC medium
mTeSR1 & SupplementsStemCell Technologies5850Animal protein-free
TeSR2 & SupplementsStemCell Technologies5860Xeno-free medium
β-mercaptoethanolSigma M7522Component of hPSC medium

MEF (CF-1) ATCC		American Type Culture Collection (ATCC) SCRC-1040For feeder culture of hPSCs
hESC-qualified MatrigelBD Bioscience354277For feeder-free culture of hPSCs
Laminin-521BioLaminaLN521-02Human recombinant protein
FGF-2 (recombinant FGF, basic)R&D Systems, MN223-FBGrowth factor in hPSC medium
CryoStor CS10StemCell Technologies7930
AccutaseInnovative Cell TechnologiesAT-1041X mixed enzymatic solution
JAK inhibitor IEMD4 Biosciences420099An inhibitor of Janus kinase
Y-27632EMD4 Biosciences688000ROCK inhibitor
Y-27632Stemgent04-0012ROCK inhibitor
Y-39983Stemgent04-0029ROCK I inhibitor
PhenylbenzodioxaneStemgent04-0030ROCK II inhibitor
ThiazovivinStemgent04-0017A novel ROCK inhibitor
BD Falcon Cell StrainerBD Bioscience35234040 µm cell strainer
Nalgene 5100-0001 Cryo 1 °CThermo Scientific C6516F-1“Mr. Frosty” Freezing Container
Lipofectamine 2000Invitrogen Inc.11668-027Transfection reagents
DharmaFECT DuoThermo ScientificT-2010-02Transfection reagent
Non-targeting miRIDIAN miRNA Transfection ControlThermo ScientificIP-004500-01-05Labeled with Dy547, to monitor the delivery of microRNAs 
SMART-shRNAThermo Scientific To be determinedLentiviral vector
pmaxGFPamaxa Inc (Lonza)Included in every transfection kitExpression plasmid for transfection control
Oct-4Santa Cruz Biotechnologysc-5279Mouse IgG2b, pluripotent marker
SSEA-1Santa Cruz Biotechnologysc-21702Mouse IgM, differentiation marker
SSEA-4Santa Cruz Biotechnologysc-21704Mouse IgG3, pluripotent marker
Tra-1-60Santa Cruz Biotechnologysc-21705 Mouse IgM, pluripotent marker
Tra-1-81Santa Cruz Biotechnologysc-21706Mouse IgM, pluripotent marker
CK8 (C51)Santa Cruz Biotechnologysc-8020Mouse IgG1, against cytokeratin 8
α-fetoproteinSanta Cruz Biotechnologysc-8399AFP, mouse IgG2a
HNF-3β (P-19)Santa Cruz Biotechnologysc-9187FOXA2, goat polyclonal antibody
Troponin T (Av-1)Thermo ScientificMS-295-P0Mouse IgG1
DesminThermo ScientificRB-9014-P1Rabbit IgG
Anti-NANOGReproCELL Inc, JapanRCAB0004P-FPolyclonal antibody 
Rat anti-GFAPZymed13-0300Glial fibrillary acidic protein
Albumin (clone HSA1/25.1.3)Cedarlane Laboratories Ltd.CL2513AMouse IgG1
Smooth muscle actin (clone 1A4)DakoCytomation IncIR611/IS611Mouse IgG2a
NestinChemicon InternationalMAB5326Rabbit polyclonal antibody
TUBB3Convance IncMMS-435PTuj1, mouse IgG2a
HNF4α (C11F12)Cell Signaling Technologies3113Rabbit monoclonal antibody
Paraformaldehyde (solution)Electron Microscopy Sciences15710PFA, fixative, diluted in D-PBS

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Human Pluripotent Stem CellsMonolayer CultureROCK Inhibitor Y 27632Laminin 521 CultureGenetic ModificationCell TransfectionStem Cell ExpansionDefined Protein CultureSmall Molecule InhibitorsColony Free Culture
Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis
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